Periodic Reporting for period 2 - NUCDDR (Nucleolar Responses to DNA Damage: rDNA, an emerging hub of genome instability)
Période du rapport: 2021-12-01 au 2023-05-31
DNA damage is a critical factor for cancer initiation and progression. The importance of chromatin environment both in susceptibility to DNA damage and efficacy of DNA break repair has gained a lot of attention over the last years. The rRNA gene repeats (rDNA) are organised in the most prominent nuclear structure, the nucleolus. rDNA repeats are spread between five different chromosomes that cluster in the nucleolus. The nucleolus is the most heavily transcribed area of the human genome with rDNA transcription accounting for up to 60% of total cellular transcription in human cells. Due to the high transcriptional activity, conflicts between the transcription and replication machineries occur with high rate in the rDNA resulting in replication stress and subsequent formation of double strand breaks (DSBs) within the loci. Formation of DSBs within the rDNA clusters affects rDNA copy numbers and leads in translocations between the five acrocentric chromosomes, often observed in cancer.
The aims of this project are:
(1) Understand how the nucleolar DNA damage response is organised. To maintain genomic stability and avoid cancer transformation cells have evolved a complex signalling network of sensor, adaptor and effector proteins that mediate DSB repair. Given the unique characteristics and the biophysical properties of the nucleolus, emerging evidence highlight that the nucleolar DNA damage response has unique features including break movement to the nucleolar exterior, transcriptional shut down and dedicated adaptor proteins that facilitate rDNA break repair. Our aim is to identify novel regulators of the nucleolar DNA damage response and unravel how the response is organised in space and time.
(2) Assess the role of R-loop formation in the nucleolar DNA damage response.
R-loops are DNA:RNA hybrids formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. Due to high transcriptional activity at the rDNA loci there is a high relevance of R-loops. Our aim is to investigate whether R-loops have a regulatory role in the organisation of the nucleolar DNA damage response.
(3) Investigate rDNA genomic instability as a driver of cancer transformation. Examine the hypothesis that rDNA becomes preferentially unstable at the early stage of cancer development and is one of the first sites that accumulates damage upon induction of replication stress (fragile site).
Conclusions of the action:
The research performed in this project resulted in additional mechanistic insights on how the DNA damage response is organised in this highly unstable nuclear sub compartment (the nucleolus) and identified a novel adaptor protein that functions specifically in rDNA DSB repair. We also created tools to study the role of RNA:DNA hybrids in rDNA break repair and provided evidence that rDNA is susceptible to replication stress and one of the first sites to accumulate damage upon oncogene induction or induction of chemically induced replication stress.
The aims of this project are:
(1) Understand how the nucleolar DNA damage response is organised. To maintain genomic stability and avoid cancer transformation cells have evolved a complex signalling network of sensor, adaptor and effector proteins that mediate DSB repair. Given the unique characteristics and the biophysical properties of the nucleolus, emerging evidence highlight that the nucleolar DNA damage response has unique features including break movement to the nucleolar exterior, transcriptional shut down and dedicated adaptor proteins that facilitate rDNA break repair. Our aim is to identify novel regulators of the nucleolar DNA damage response and unravel how the response is organised in space and time.
(2) Assess the role of R-loop formation in the nucleolar DNA damage response.
R-loops are DNA:RNA hybrids formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. Due to high transcriptional activity at the rDNA loci there is a high relevance of R-loops. Our aim is to investigate whether R-loops have a regulatory role in the organisation of the nucleolar DNA damage response.
(3) Investigate rDNA genomic instability as a driver of cancer transformation. Examine the hypothesis that rDNA becomes preferentially unstable at the early stage of cancer development and is one of the first sites that accumulates damage upon induction of replication stress (fragile site).
Conclusions of the action:
The research performed in this project resulted in additional mechanistic insights on how the DNA damage response is organised in this highly unstable nuclear sub compartment (the nucleolus) and identified a novel adaptor protein that functions specifically in rDNA DSB repair. We also created tools to study the role of RNA:DNA hybrids in rDNA break repair and provided evidence that rDNA is susceptible to replication stress and one of the first sites to accumulate damage upon oncogene induction or induction of chemically induced replication stress.
DNA lesions occur across the genome and constitute a threat to cell viability; however, damage at specific genomic loci has a relatively greater impact on overall genome stability. The heavily transcribed ribosomal RNA (rDNA) repeats that give rise to the ribosomal RNA are clustered in a unique chromatin structure, the nucleolus (reviewed in Kasselimi et al, 2022 Trends in Biochemical Sciences). Due to its highly repetitive nature and transcriptional activity, the nucleolus is considered a hotspot of genomic instability. Employing targeted double strand breaks in the rDNA we uncovered RASSF1A, a tumour suppressor adaptor protein that is commonly inactivated in cancer as a novel regulator of the nucleolar DNA damage response. We found that RASSF1A gets recruited at rDNA double strand breaks (DSBs) via 53BP1 and facilitates 53BP1 function in local ATM signal amplification. RASSF1A loss of expression, a common event in cancer, results in persistent rDNA breaks, sensitivity in rDNA damage and discrepancies in rDNA copy numbers in lung adenocarcinoma patient cohorts (Tsaridou et al., 2022 EMBO Reports).
Additionally, we developed tools (innovative imaging-based approaches and stable cell lines) to study how the rDNA damage response is regulated in time and space. We employed cell systems where we induced oncogene expression or induced chemically replication stress to explore rDNA as a fragile site. Our data so far supports that rDNA is one of the first genomic sites to accumulate DNA lesions upon induction of replication stress highlighting its contribution to genomic instability in cancer.
Additionally, we developed tools (innovative imaging-based approaches and stable cell lines) to study how the rDNA damage response is regulated in time and space. We employed cell systems where we induced oncogene expression or induced chemically replication stress to explore rDNA as a fragile site. Our data so far supports that rDNA is one of the first genomic sites to accumulate DNA lesions upon induction of replication stress highlighting its contribution to genomic instability in cancer.
We have contributed to the overall understanding of how the nucleolar DNA damage response is organised and have uncovered a novel regulator of the response. Our data suggests that RASSF1A tumor suppressor is specifically recruited to ribosomal DNA (rDNA) breaks promoting local establishment of ATM signal at damaged nucleoli. Selective recruitment at rDNA break sites, highlights the necessity of additional mechanisms to secure high fidelity DNA repair at sites where break repair may be more challenging. RASSF1A expression is often lost in several tumour types including lung cancer and has been proposed as an attractive biomarker. Our findings have translational value as they highlight how epigenetic inactivation of the scaffold contributes in genomic instability during malignant transformation and further support the use of RASSF1A methylation as a potential biomarker for patient therapy stratification (eg. explore the impact of RASSF1A methylation status in patient treatment with Polymerase I inhibitors that are currently in clinical trials).